25-hydroxy vitamin d3 derivatives, process for their production, and assay method using the same

ABSTRACT

A novel 25-hydroxy vitamin D 3  amino acid derivative of the formula ##STR1## wherein R 1  is C 1-10  alkylene, is produced by removing a protective group for the amino group, e.g. 9-fluorenylmethyl-oxycarbonyl, in the presence of a base in an inert solvent. A radioisotope iodine-labeled residue is then attached to the amino group to produce a derivative useful in the assay of 25-hydroxy vitamin D 3  in a specimen.

FIELD OF THE INVENTION

This invention relates to novel 25-hydroxy vitamin D₃ derivatives, theirproduction and an assay method using the same.

DESCRIPTION OF THE PRIOR ART

For the assay of 25-hydroxy vitamin D₃, a competitive protein bindingassay (CPBA) method and a radio immunoassay (RIA) method are known. Inthese methods, a tritium [³ H]-labeled compound is used (PCT Open No.56-500538). An example thereof is a tritium-labeled 25-hydroxy vitaminD₃ derivative with position-1 carbon (Japan Pat. Unexam. Publ. No.60-163859). As the vitamin D binding protein used in the CPBA method, itis also known to use the plasma of rats fed with vitamin D-deficientfeed [Vitamin, 55(12), 595-605: 1981]. As haptens for the preparation ofantibodies used in the RIA method, substances having carbonyl in a sidechain terminal are known (Japan Pat. Unexam. Publ. Nos. 58-92656,55-47653 and 59-148775 )

In these assay methods, as the isotopically labeled compound, tritium [³H]-labeled 25-hydroxy vitamin D₃ is used.

In the assay of 25-hydroxy vitamin D₃ in a specimen, the prior-usedtritium [³ H]-labeled compounds were of low specific activity ascompared with either ³² P or ¹²⁵ I as to radiation energy and sufferedfrom high cost and involved cumbersome operations.

BRIEF SUMMARY OF THE INVENTION

We have discovered radioisotope iodine-labeled 25-hydroxy vitamin D₃derivatives with high radiation energy and superior characteristics ofuse.

Heretofore, there have been known isotopically labeled vitamin D₃derivatives with tritium [³ H] labeling, which have β-ray nuclearemission with low radiation energy per unit time.

However, no information was known on ¹²⁵ I-labeled vitamin D₃derivatives which have γ-ray nuclear emission with high radiationenergy, because the conjugated triene structure of vitamin D₃ isunstable and was thought to be subject to auto-degradation by inducingradical reactions.

The ¹²⁵ I-labeled 25-hydroxy vitamin D₃ derivatives of the presentinvention are, on the contrary, stable without structural degradation byγ-rays and hence are useful for radio immunoassay.

In radioisotope labeling, direct labeling (chloramin-T method) andindirect labeling (Bolton-Hunter reagent method) are known. (AmershamNote, 1 Nov., 1981).

The indirect method under mild reaction conditions is preferable due tothe instability of vitamin D in the presence of acid, oxygen, oxidizingagents, heat or light.

We have synthesized novel 25-hydroxy vitamin D₃ amino acid derivativeshaving a terminal amino group in a side chain. The said derivatives ashaptens are bound with carrier protein and are inoculated into animalsto obtain antibodies. Furthermore, the derivatives are labeled withradioisotope iodine. Using the obtained antibodies and radioisotopeiodine-labeled compounds, we have established a radio immunoassaysystem, then have found that in an assay of 25-hydroxy vitamin D₃ in aspecimen the said assay system is highly sensitive and useful.

Also in the CPBA method using DBP, radioisotope iodine-labeled compoundsof the present invention are found to be useful for assaying 25-hydroxyvitamin D₃ with high sensitivity.

OBJECTS OF THE INVENTION

An object of the present invention is to provide novel 25-hydroxyvitamin D₃ amino acid derivatives of the formula ##STR2## wherein R₁ isC₁₋₁₀ alkylene.

Another object of the present invention is to provide a process for theproduction of novel 25-hydroxy vitamin D₃ amino acid derivatives [I],which comprises removing a protective group for the amino group in25-hydroxy vitamin D₃ derivatives of the formula ##STR3## wherein R₁ isC₁₋₁₀ alkylene and R₂ is a protective group for the amino group, in thepresence of a base in an inert solvent.

A further object of the present invention is to provide radioisotopeiodine-labeled 25-hydroxy vitamin D₃ derivatives of the formula ##STR4##wherein R₁ is C₁₋₁₀ alkylene and R₃ is a radioisotope iodine-labeledresidue.

A still further object of the present invention is to provide an assaymethod for 25-hydroxy vitamin D₃ in a specimen, which comprises thefollowing steps:

adding to a specimen containing 25-hydroxy vitamin D₃ a radioisotopeiodine-labeled 25-hydroxy vitamin D₃ derivative of formula [III];

adding to the mixture thus formed anti-25-hydroxy vitamin D₃ antibody;

separating the thus-generated bound form of radioisotope iodine-labeled25-hydroxy vitamin D₃ derivative-anti 25-hydroxy vitamin D₃ antibody andunreacted free form of radioisotope iodine-labeled 25-hydroxy vitamin D₃derivative; and

measuring the amount of labeled radioisotope iodine in a bound or freeform.

Still another object of the present invention is to provide an assaymethod for 25-hydroxy vitamin D₃ in a specimen, which comprises thefollowing steps:

adding to a specimen containing 25-hydroxy vitamin D₃ a radioisotopeiodine-labeled 25-hydroxy vitamin D₃ derivative of formula [III];

adding to the mixture thus formed vitamin D binding protein (hereinafterdesignated as DBP);

separating the thus-generated bound form of radioisotope iodine labeled25-hydroxy vitamin D₃ derivative-DBP and unreacted free form ofradioisotope iodine labeled 25-hydroxy vitamin D₃ derivative; and

measuring the amount of labeled radioisotope iodine in a bound of freeform.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying drawings

FIG. 1 is the standard curve of an [¹²⁵ I]-labeled 25-hydroxy vitamin D₃derivative;

FIG. 2 is the standard curve of an [¹²⁵ I]- or [³ H]-labeled 25-hydroxyvitamin D₃ derivative;

FIG. 3 is the standard curve of a monoiodo [¹²⁵ I]- or diiodo [¹²⁵I]-labeled 25-hydroxy vitamin D₃ derivative;

FIG. 4 is the standard curve of compound [IV] wherein R₁ is [--(CH₂)n--]and n=1, n=2 and n=3;

FIG. 5 is the standard curve of an [¹²⁵ I]-labeled 25-hydroxy vitamin D₃derivative by the CPBA method using DBP instead of anti-25-hydroxyvitamin D₃ antibodies; and

FIG. 6 is the stability curve of an [¹²⁵ I]-labeled 25-hydroxy vitaminD₃ derivative by the CPBA method using DBP instead of anti-25-hydroxyvitamin D₃ antibodies.

DETAILED DESCRIPTION OF THE INVENTION

25-Hydroxy vitamin D₃ amino acid derivatives of formula [I] can beobtained by covalently bonding with an amino acid through the 3β-hydroxygroup of 25-hydroxy vitamin D₃. In formula [I], R₁ is C₁₋₁₀ alkylene.Compound [I] can be used a derivative of a hapten in the preparation ofanti-25-hydroxy vitamin D₃ antibodies. The linkage of a haptenderivative and a carrier protein is preferably a 1-10 carbon chain, andmore preferably 2-6 carbon, so R₁ is more preferably C₂₋₆ alkylene.

Compound [I] can be prepared by first reacting 25-hydroxy vitamin D₃with an amino acid having a protected amino group to obtain a 25-hydroxyvitamin D₃ derivative [II] and removing the protective group therefrom.

The amino acid is a compound of the formula

    HOOC--R.sub.1-- NH.sub.2                                   [IV]

wherein R₁ has the same meaning as hereinbefore.

Examples of alkylene R₁ are n=1; glycine, n=2; β-alanine, n=3;γ-amino-butyric acid and β-amino-isobutyric acid, n=4; δ-amino valericacid, n=5; ε-amino-n-caproic acid, n=6; 7-amino heptanoic acid, andn=10; 11-amino undecanoic acid. The carbon chain in the amino acid ispreferably a straight chain and is preferably C₂₋₆ alkylene.δ-amino-levulinic acid, glycylglycine or δ-amino acid, i.e. D and/orL-lysine can also be used. An amino acid having side chain hydroxyl, forexample 4-amino-3-hydroxybutyric acid can be used if the hydroxyl groupis protected.

The protective group for the amino group may be a group which can easilybe removed under mild conditions and under weakly basic conditions dueto the possible instability of vitamin D under severe removalconditions. Examples are 9-fluorenyl methyloxycarbonyl (hereinafterdesignated as Fmoc), 9-(2-sulfo)-fluorenyl methyloxycarbonyl,1,1-dimethyl-2-cyanoethyloxy carbonyl and 5-benzisoxazolylmethyloxycarbonyl. 9-Fluorenyl methyloxycarbonyl is preferred. Aminogroup protection can be effected by known methods therefor, by reactingan activated derivative, for example an activated ester having Fmoc,such as 9-fluorenylmethyl -succinimidylcarbonate or9-fluorenylmethylchloroformate, with an amino acid [L. A. Carpino and G.Y. Ham, J. Oro. Chem. 37: 3404 (1972)].

25-hydroxy vitamin D₃ amino acid derivatives of formula [II] can beobtained, for example using an Fmoc-amino acid as an amino protectedamino acid, by reacting one equivalent of 25-hydroxy vitamin D₃ with oneequivalent of Fmoc-amino acid or its acid anhydride, acid halide oractivated ester to obtain compound [II]. More preferably, one equivalentof Fmoc-amino acid is reacted with one equivalent of mixed anhydridehaving another acid, under an inert gas, and a base in an anhydroussolvent. The reason for reacting the said mixed anhydride with25-hydroxy vitamin D₃ is to prevent the formation of a by-product,25-hydroxy vitamin D₃ having an Fmoc-amino acid on a hydroxy group atposition-25.

Examples of other acids in a mixed anhydride are valeric acid, pivalicacid or isobutylchloroformate. Pivalic acid is preferred. Examples ofanhydrous solvents are anhydrous organic solvents such astetrahydrofuran or dioxane. Examples of bases are preferablydimethylaminopyridine (DMAP) and piperidinopyridine (PPY), which arepreferred for the esterification of secondary or tertiary alcohols withstearic hindrance. The preferred inert gases are argon and nitrogen. Thereaction proceeds preferably at 0-20° C. for 1-3 hours. Thethus-obtained 25-hydroxy vitamin D₃ derivative [II] can be purified, ifnecessary, by any conventional purification method such as columnchromatography or thin layer chromatography (TLC).

The said 25-hydroxy vitamin D₃ derivative of formula [II] is subjectedto de-protection of the amino group, i.e. removal of Fmoc, in thepresence of a base in an inert solvent to produce a 25-hydroxy vitaminD₃ derivative of the formula [I]. Examples of bases are piperidine,morpholine or ethanolamine. Morpholine is preferred. Examples of inertsolvents are ethanol and methanol, preferably anhydrous ethanol oranhydrous methanol. The reaction proceeds under an inert gas in the darkat 0-20° C. for 1-3 hours.

The thus-obtained 25-hydroxy vitamin D₃ derivative [I] can be purifiedby column chromatography or TLC.

In a radioisotope iodine-labeled 25-hydroxy vitamin D₃ derivative offormula [III], R₁ has the same meaning as in formula [I] and R₃ is aradioisotope iodine-labeled residue. Examples of radioisotopes of iodineare ¹²⁵ I and ¹³¹ I. The relatively long-half-life isotope ¹²⁵ I ispreferred. Examples of radioisotopes of iodine-labeled residues are3-(4-hydroxy-3-iodo [¹²⁵ I] phenyl)-propionyl, 3-(3,5-diiodo [¹²⁵I]-4-hydroxyphenyl) propionyl, 2-(4-hydroxy-3-iodo [¹²⁵ I] phenyl)acetyl, 2-(3,5-di-iodo [¹²⁵ I]-4-hydroxyphenyl) acetyl, 2-iodo [¹²⁵ I]acetyl, 4-iodo [¹²⁵ I] benzoxymethyl carbonyl and N-substituted-3-iodo[¹²⁵ I] tyrosine residue. Among these, 3-(4-hydroxy-3-iodo [¹²⁵ I]phenyl) propionyl and 3-(3,5-diiodo [¹²⁵ I]-4-hydroxyphenyl propionylare preferred.

In the production of radioisotope iodine-labeled 25-hydroxy vitamin D₃derivatives, a 25-hydroxy vitamin D₃ amino acid derivative [I] islabeled with radioisotope iodine by an indirect labeling method by meansof the Bolton-Hunter reagent to obtain a radioisotope iodine-labeled25-hydroxy vitamin D₃ derivative. An indirect labeling method herein isa method for producing radioisotope iodine-labeled 25-hydroxy vitamin D₃derivative [III] by reacting a reactive derivative [IV] hereinbelow,which has a radioisotope iodine-labeled residue, with 25-hydroxy vitaminD₃ amino acid derivative [I].

The above reactive derivative [IV] is expressed by the formula

    R.sub.3-- X                                                [IV]

wherein R₃ has the same meaning as hereinbefore, and X issuccinimidyl-N-oxy, phthalimidyl-N-oxy,5-norbornene-2,3-dicarboximidyl-N-oxy or maleimidyl-N-oxy.N-succinimidyl-3-(4-hydroxy-3-iodo [¹²⁵ I] phenyl) propionate, for whichR₃ is 3-(4-hydroxy-3-iodo [¹²⁵ ] phenyl) propionyl and X issuccinylimidyl-N-oxy in the compound [IV], is a commercially available[¹²⁵ I] Bolton-Hunter reagent. For example, an indirect labeling methodon 25-hydroxy vitamin D₃ amino acid derivative [I] using the saidBolton-Hunter reagent can be conducted by reacting several μ moles toseveral m mole of radioisotope iodine [¹²⁵ I] Bolton-Hunter reagent witha 500-2,000-fold excess amount, preferably 1000-fold excess amount of25-hydroxy vitamin D₃ amino acid derivative, at 0-30° C. for 12-72hours. In order to increase the effect on the RIA or CPBA method, theproduced radioisotope-labeled 25-hydroxy vitamin D₃ derivative [III] ispreferably purified by TLC or HPLC. The thus-obtained ¹²⁵ I-labeled25-hydroxy vitamin D₃ derivative is stable at -20° C. in ethanol formore than the two month half-life of ¹²⁵ I, and can be used in radioimmunoassays.

The storage temperature is preferably as low as possible, e.g. 5°C.--20° C., but preferably even below -20° C., in alcohol or ether,under an inert gas.

Anti-25-hydroxy vitamin D₃ antibodies can be prepared by inoculating aconjugation of a hapten, i.e. 25-hydroxy vitamin D₃ amino acidderivative [I] and a carrier protein into an animal. Examples of carrierproteins which are essential for obtaining an immunogenic antigen for ahapten, are simple proteins, polypeptides and complex proteins such asglycoproteins. Examples of simple proteins are bovine serum albumin(BSA), human serum albumin or human serum globulin. An examples of apolypeptide is polylysine. An example of a glycoprotein is mucoprotein.Among these, simple proteins are preferred, and bovine serum albumin andhuman serum albumin are the most preferred.

25-Hydroxy vitamin D₃ amino acid derivative [I] and a carrier proteinare covalently bonded in the presence of a condensation reagent orcrosslinkage reagent. Examples of condensation reagents or crosslinkagereagents are dicyclohexylcarbodiimide (DCC), acid anhydride andglutaraldehyde. DCC is preferred. The conjugation radio of the25-hydroxy vitamin D₃ amino acid derivative and the carrier protein canbe, due to the decreasing titer of the antibodies if in excess 10-40,molecules of 25-hydroxy vitamin D₃ amino acid derivative per onemolecule of carrier protein.

The thus-prepared conjugate for antibody production is inoculated intoan animal to produce antibodies. Inoculation can be by parenteraladministration such as subcutaneous or intracutaneous injection. Forinoculation, a conjugated antigen of the above 25-hydroxy vitamin D₃amino acid derivative-carrier protein is dissolved in a buffer solutionor physiological saline, together with an equal amount of completeFreund's adjuvant (C.F.A.) The mixture is emulsified completely, andinoculated subcutaneously or intracutaneously into a homeothermalanimal, about ten times in every 1-3 weeks to immunize the same.Alternatively, the conjugated antigen can be directly inoculated into aspleen. During the immunization period, the serum antibody titer ismeasured at constant time intervals; and at maximum titer, a whole bloodsample is collected and allowed to stand for coagulation. The coagulatedsample is centrifugally separated to obtain an antiserum containinganti-25-hydroxy vitamin D₃ antibodies.

The type of homeothermal animals is not limited and can be any animalwhich has antibody production activity. To obtain large amounts ofantibodies, sheep or bovines can be used. In general, rabbits or ratsare preferred.

The isolation of anti-25-hydroxy vitamin D₃ antibody from the antiserumcan be performed by any conventional method for antibody purification.For example, ammonium sulfate fractionated antiserum can be treated byion-exchange chromatography or gel-filtration.

Another method of the production of the antibodies is that spleen cells,which can produce the desired antibodies of the animal, are inooulatedwith conjugated antigen of 25-hydroxy vitamin D₃ amino acidderivative-carrier protein, and are fused with established myelomacells, and the thus-obtained hybridoma is cultured Then the monoclonalantibodies produced by the said hybridoma are used.

For example, an emulsion, which is prepared by mixing a conjugatedantigen of 25-hydroxy vitamin D₃ amino acid derivative-carrier proteindissolved in buffer solution or physiological saline and an equal amountof C.F.A., is inoculated subcutaneously in mice, for example Balb/c forsensitization. Cell-fusion is performed 3-5 days after finalsensitization. At 3-5 days after final sensitization, spleen cells whichproduce anti-25-hydroxy vitamin D₃ antibodies are collected and fusedwith established myeloma cells which can be cultured for a long term.Long-term culturable established cells may be defined as cells which canbe cultured and grown for a long term in vitro or in vivo, and which canproduce immunoglobulin or its related proteins. Generally, well-grownmyeloma cells are used. Preferred examples of myeloma cells are thecell-lines P3-NSI/1-Ag4-1, P3-X63-Ag8UI, SP2/U-Ag14 andMPCIl-45.6.TG.1.7. In the present invention, P3-X63-Ag8UI is preferred.The cells can be cultured in a conventional cell culture medium. Forexample culture can be performed in a medium of 10% FCS to which isadded RPMI 1640 (tradename, Flow Laboratory) to which is addedglutamine, pyruvic acid, penicillin and streptomycin. For stock culture,S-azaguanine is added to the above medium. Myeloma cells 1-3×10⁸ cells,are used for cell fusion. Spleen cells can be prepared by cutting mousespleen and crushing it on a mesh to prepare a spleen cell suspension.Washed cells, generally 1-3×10⁸ cells, are fused with myeloma cells bymixing together The ratio of mixed cells can be myeloma cells:spleencells 1:3-10. Cell fusion is achieved in a medium for hybridoma. In thecell fusion, a conventional cell-fusion method using a promoter such asSendai virus or polyethylene glycol (PEG) is preferred. PEG isparticularly preferred.

The fused cells are inoculated into the medium for hybridoma andincubated, then selected by incubating in HAT medium. HAT medium is amedium for hybridoma with added hypoxanthine, aminopterin and thymidine.Since more than two hybridomas can be grown in the well ofcell-separation plate, more than two kind of antibodies are possiblyproduced, or no-antibody producing cells may be a contaminant; so inorder to obtain cells having the same properties, each clone should beseparated. For cloning, a limiting dilution culture or soft agar cultureis used. In this invention, a limiting dilution culture is preferred.

The thus-obtained hybridoma secreting anti-25-hydroxy vitamin D₃antibodies with high titer can be stored after lyophilization at anearly stage. Lyophilization can be performed by a conventional method,namely cell suspension in a small tube or ampule with freezing in a -80°C. freezer and storage in liquid nitrogen. Another example of hybridomaproduction is that the above hybridoma is inoculated intraperioneally inpristan (2,6,10,14-tetramethylpentadecane, Aldrich Chemicals) treatedmice, and after about 10 days the ascites are collected. Another methodis that the hybridoma is incubated into bovine fetal serum with addedRPMI medium or into Darbecco-modified Eagle medium The antibodies thusobtained can be purified by any conventional method For example, theantibodies are fractionated with ammonium sulfate and treated byion-exchange chromatography, gel filtration and affinity chromatographyto fractionate IgG. Then purified anti-25-hydroxy vitamin D₃ monoclonalantibodies can be obtained.

Furthermore, anti-25-hydroxy vitamin D₃ monoclonal antibody-producingcells can b inoculated and grown in an animal having identicalhistocompatibility antigens or in nude mice as a tumor, and the growncells are collected and the monoclonal antibodies separated therefrom.

In an assay of 25-hydroxy vitamin D₃, anti-25-hydroxy vitamin D₃polyclonal antibodies or anti-25-hydroxy vitamin D₃ monoclonalantibodies (hereinafter sometimes collectively designatedanti-25-hydroxy vitamin D₃ antibody) can be used in its soluble state orin an immobilized state. An insoluble carrier and anti-25-hydroxyvitamin D₃ antibodies are bound by using a polyfunctional reagent andthe immobilized antibody has antibody titer against 25-hydroxy vitaminD₃. Examples of polyfunctional reagents are compounds having more thantwo groups which can react with functional groups such as amino,hydroxyl, carboxyl and thiol, and comprise aldehydes such assuccinaldehyde, glutaraldehyde and adipoaldehyde; dicarboxylates such asmalonic acid, succinic acid glutaric acid or adipic acid or theirreactive derivatives; diisocyanates such as hexamethylene diisocyanateor 2,4-toluenediisocyanate; diisothiocyanates such as hexamethylenediisothiocyanate; maleimide carboxylates such as maleimide benzoate ormaleimide phenylacetate or their functional derivatives; dimaleimidessuch as N,N -ethylene-bis-maleimide or N,N'-0-phenylene dimaleimide,bisdiazobenzidine, diethylmalonimidate, dimethyladipinimidate orN,N'-polymethylene-bisiodo acetamide and thiocarboxylates such as3-(2'-benzothiozolyl-dithio) propionate and 3-(2'-pyridyldithio)propionate or their functional derivatives. The polyfunctional reagentcan be selected by considering the bonding of functional groups such asamino, carboxyl, hydroxyl or thiol in anti-25-hydroxy vitamin D₃antibody.

The immobilized carrier is a carrier having a reactive group which doesnot bond with groups for bonding with antibodies in polyfunctionalgroups. Examples of immobilized carriers are insoluble proteins such asalbumin or gelatin; epichlorhydrin-treated insoluble polysaccharidessuch as agarose, cellulose or dextrin; insoluble polymers or copolymersof acrylonitrile, acrylic acid, acrylate ester, methacrylic acid,methacrylate ester, vinyl alcohol, binyl acetate, styrene, aminostyrene,chlorstyrene, maleic acid and fumaric acid, which is treated withbromocyanate and is introduced with a spacer corresponding to aminogroup introduction, and an insoluble inorganic carrier which isintroduced with a functional group such as an amino group into aninorganic compound such as one of silicon and aluminum The immobilizedcarrier may also be a carrier which can bind anti-25-hydroxy vitamin D₃antibodies by physical adsorption.

The immobilized carrier is preferably in particulate form which can beeasily isolated by filtration, for example beads having a diameter morethan 1 mm and preferably more than 5 mm, or a spindle form whichcorresponds to the bottom shape of an antigen-antibody reaction tube.

The introduction of reactive group into anti-25-hydroxy vitamin D₃antibodies using a spacer-introducing reagent can be performed byintroducing additional functional groups such as aldehyde, carboxyl,amino or thiol, reacting with at least one spacer introducing reagent,for example a dialdehyde such as succinaldehyde, glutaraldehyde oradipoaldehyde; a reactive derivative such as acid chloride, asuccinimide ester or p-nitrophenyl ester of ω-amino butyric acid orω-amino glutamic acid; a reactive derivative of dicarboxylic acids suchas malonic acid, succinic acid, glutaric acid or adipic acid; diaminessuch as hexamethylene diamine or decamethylene diamine; reactivederivatives of 3-(2'-pyridyl-dithio) propionic acid or3-(2'-benzothiazolyldithio) propionic acid, S-acetyl-mercapto succinicanhydride or thiols such as 2-aminoethanethiol.

Anti-25-hydroxy vitamin D₃ antibodies are condensed directly or throughpolyfunctional reagents with reactive groups in the immobilized carrier.Condensation reactive process generally at 0-40° C. in a pH 6.0-8.5buffer solution or organic solvent or mixture thereof Furthermore, asecond antibody which is obtained by immunizing large mammals inoculatedwith an immunoglobulin fraction in a serum, which is used for antibodyproduction of 25-hydroxy vitamin D₃, is immobilized and anti-25-hydroxyvitamin D₃ antibody is bound thereto by an antigen-antibody reaction toprepare the immobilized antibody.

The thus-obtained immobilized antibodies are washed and stored.

In an assay of 25-hydroxy vitamin D₃ in a specimen using anti-25-hydroxyvitamin D₃ antibodies in a liquid soluble phase, a fixed amount ofradioisotope iodine-labeled 25-hydroxy vitamin D₃ derivative [III] isfirst added to a specimen containing 25-hydroxy vitamin D₃, then anoptimum amount of anti-25-hydroxy vitamin D₃ antibodies is added to forman antigen antibodies reaction product.

The thus-formed labeled 25-hydroxy vitamin D₃ derivative-anti-25-hydroxyvitamin D₃ antibodies binding complex, and the 25-hydroxy vitamin D₃-antibodies binding complex, and the free labeled 25-hydroxy vitamin D₃derivative are separated by using specific antibodies foranti-25-hydroxy vitamin D₃ antibodies. The said specific antibodies arehereinafter referred to as second antibodies. The second antibodies canbe obtained, for example by inoculating a normal immunoglobulin fractioninto the serum of an animal which is used for antibodies production of25-hydroxy vitamin D₃, as an antigen to immunize, then isolating thesecond antibodies from the thus-obtained anti-serum. The secondantibodies can be purified if necessary by any known method, or they canpreferably be used in the condition of an antiserum.

An assay method for 25-hydroxy vitamin D₃ in a specimen usinganti-25-hydroxy vitamin D₃ antibodies and radioisotope iodine-labeled25-hydroxy vitamin D₃ derivative is illustrated as follows:

25-Hydroxy vitamin D₃ in a specimen such as a known serum, is extractedfrom the serum specimen. A sample, which is a mixture of serum and anequal amount of added solvent, is stirred allowed to stand andcentrifuged. The separated supernatant solution is treated by columnchromatography and a fraction of 25-hydroxy vitamin D₃ is collected andpreferably is purified by HPLC.

The 25-hydroxy vitamin D₃ fraction can be checked by previously addedtritium [³ H]-labeled 25-hydroxy vitamin D₃. The 25-hydroxy vitamin D₃fraction is dried in vacuo, flushed with argon gas and dissolved inethanol to prepare a specimen. A fixed amount of radioisotopeiodine-labeled 25-hydroxy vitamin D₃ derivative is added to thespecimen, and the most suitable amount of anti-25-hydroxy vitamin D₃antibody is added thereto. The mixture is incubated in a medium forantigen-antibodies such as a phosphate buffer or a veronal buffer at4-5° C. for about 15-72 hours to promote the competitive reaction ofradioisotope iodine-labeled hapten and non-labeled hapten to antibodies.The thus-formed antigen antibody binding complex, namely a bound form ofradioisotope iodine-labeled 25-hydroxy vitamin D₃derivative-anti-25-hydroxy vitamin D₃ antibody (B) and an unreacted freeform of radioisotope iodine-labeled 25-hydroxy vitamin D₃ derivative (F)are separated by the dextran-charcoal (DC) method by filtration or bycentrifugation at 3,000 r.p.m. for 15 mins. Upon B-F separation, theradioactivity of each of B and F is measured.

The amount of 25-hydroxy vitamin D₃ (H) in a specimen is calculated bymeasuring the radioactivity B/(B +F) or B/F. Namely, when the amount ofH is increased, the radioactivity of B is decreased and that of F isincreased. Therefore, the unknown amount of H can be determined bymeasuring the radioactivity of B and F from a previously plottedstandard curve of known amounts.

Upon B-F separation, when the double antibody technique is used withsoluble state antibodies, second antibodies, preferably secondantibodies containing an antiserum and if necessary a normal serum ofthe same kind of animal used for anti-25-hydroxy vitamin D₃ antibodiesproduction, are added after the competitive reaction and incubated for1-12 hours. Thereafter the formed binding complex is precipitated bycentrifugation at 3,000 r.p.m. for 10-30 mins. to separate precipitate(B) and supernatant (F), then the radioactivity of B or F is measured.

According to the present assay method, a standard curve for 2pg/Test-256 pg/Test can be prepared, and a rapid reaction time of 16hours at 5 C or 1 hour at 37° C. can be achieved. Furthermore, theoperation after the reaction is quite simple. Moreover, dilution andrecovery tests using the specimens display linearity with highprecision. The correlation coefficient between the [¹²⁵ I] RIA method ofthe present invention and the known [³ H] CPBA method upon assay of25-hydroxy vitamin D₃ in human serum is correlation coefficient γ=0.980(y=0.928X+1.90, n=18).

Also when antibodies are replaced by known DBP, it can be reacted with aradioisotope iodine [¹²⁵ I]-labeled 25-hydroxy vitamin D₃ derivative anda standard curve for the range 1-32 pg/Test can be prepared. The DBP canbe obtained from the serum of chickens, rats, mice, rabbits, goats,sheep, bovines or humans.

The following examples illustrate the present invention but are not tobe construed as limiting:

REFERENTIAL EXAMPLE 1

Extraction and purification of 25-hydroxy vitamin D₃ in serum:

A mixture of serum 0.5 ml and acetonitrile 0.5 ml was stirred in aBORTEX miser and then allowed to stand for 30 mins. The supernatantsolution (0.8 ml) obtained by centrifugation at 3,000 r.p.m. for 10mins. was charged on a Sept-pack C-18 cartridge column (tradename,MILLIPORE, Waters Corp.) which was activated with ethanol andequilibrated with 50% acetonitrile, and eluted with hydratedacetonitrile The column was further washed with 50% acetonitrile (4 ml)then eluted with 64% acetonitrile (4 ml) (a fraction of 1α,25-dihydroxyvitamin D₃), and eluted with 73% acetonitrile (4 ml) (a fraction of25-hydroxy vitamin D₃ and 24,25-dihydroxy-vitamin D₃). The fractioneluted with 73% acetonitrile was dried in vacuo and flushed with argongas to obtain a fraction containing 25-hydroxy vitamin D₃. Thethus-obtained crude fraction was dissolved in a mixture (200 μl) ofn-hexane-isopropanol (9:1) and purified by HPLC in a Zorbax-SIL (DupontInc.) 0.46×25 cm column. The above fractions of 25-hydroxy vitamin D₃was checked by previously added tritium [³ H]-labeled 25-hydroxy vitaminD₃ [ 26,27-methyl-³ H]. The fraction of 25-hydroxy vitamin D₃ (Rt=4-5mins.) was dried in vacuo and flushed with argon gas.

The said fractions were dissolved in ethanol (2 ml) and 20 μl thereofwas used for an assay.

The recovery of 25-hydroxy vitamin D₃ was 94.6±2.4% (n=18).

REFERENTIAL EXAMPLE 2

3-(N-fluorenyl methyloxy carbonyl) amino propionic acid:

γ-amino-n-propionic acid (178 mg, 2mM) was added toN-succinimidyl-9-fluorenylmethyloxycarboxylate (674 mg, 2mM) dissolvedin a mixture of tetrahydrofuran (THF) -dimethylformamide (DMF)-H₂ O(1:2:2) (25 ml) and the mixture was reacted at room temperatureovernight. The reaction solvent was distilled off in vacuo and theresidue was charged on a column of silica gel (Wako-gel C-200, 75 g) andseparated and purified by eluting with CHC13:methanol=9:1 to obtain theabove oompound (538.8 mg) (Yield: 86.5%).

NMR. δ (DMSO-d6);

2.50-2.60 (2H, t, --CH₂₋₋ Co--);

3.28-3.35 (2H, m, --CH₂₋₋ N--);

4.33-4.44 (3H, m, Fmoc);

7.35-8.06 (8H, m, Fmoc).

EXAMPLE 1

25-hydroxy vitamin D₃ -3β-0-3 [3'-(N-9-fluorenylmethyloxy carbonyl)aminopropionate]:

Pivaloyl chloride (7.38 μl, d=0.979, 0.06 mM) and dimethylamino pyridine(DMAP, 7.32 mg, 0.06 mM) were added to 3-(N-9-fluorenyl methyloxycarbonyl) amino propionic acid (18.7 mg, 0.06 mM) dissolved in dry THF(3 ml), and the mixture was reacted at -15° C. for 15 mins. under anargon atmosphere in the dark. A THF solution (1 ml) of 25-hydroxyvitamin D₃ (24.0 mg, 0.06 mM) was added thereto and the mixture wasreacted at 0° C. for 1 hour and at room temperature for 1 hour,whereupon 25-hydroxy vitamin D₃ disappeared. Methanol (0.5 ml) was addedto the mixture to stop the reaction, then distilled off in vacuo. Theresidue was purified by preparative TLC [Art. 5717, Merck, 20×20 cm,developer: ethylacetate-hexane (1:2)] to obtain the compound (30.1 mg)(yield: 72.4%).

NMR. δ (CDCl₃) ppm;

0.537 (3H, s, CH₃ -18);

3.30-3.60 (2H, m, --CH₂₋₋ N--);

4.10-4.50 (3H, m, Fmoc);

4.80-5.60 (4H, m, H-19E, H-3α, H-19Z, --NH);

5.94-6.29 (2H, m, H-7, H-6);

7.20-7.81 (8H, m, Fmoc).

UV. λ^(EtOH) _(max) nm (300.2, 266.4, 214.4

EXAMPLE 2

25-hydroxy vitamin D₃ -3β-0-(3-aminopropionate):

25-Hydroxy vitamin D₃ -3β-0-[3-(N-9'-fluorenyl methyloxy carbonyl)aminopropionate] (30.1 mg, 0.043 mM) obtained in Example 1 was dissolvedin ethanol (1 ml). Morpholine (10 ml) was added thereto and the mixturewas stirred at room temperature for 1 hour, under an argon atmosphere inthe dark, to complete the reaction. The reaction mixture wasconcentrated in vacuo and purified by preparative TLC [Art. 5717, Merck,10×20 cm, developer: ethyl acetate-MeOH (1:4)] to obtain the compound(8.3 mg) (yield: 40.6%).

Ninhydrin colorization: positive

NMR. δ (CDC1₃) ppm;

0 54 (3H, s, CH₃ -18);

2.36-2.52 (2H, m, --CO--CH₂₋₋);

2.90-3.05 (2H, m, --CH₂₋₋ N--);

3.50-4.00 (2H, b, --NH₂);

4.86-5.08 (3H, m, H-19E, H-3α, H-19Z);

5.90-6.30 (2H, m, H-7, H-6).

UV. λ^(EtOH) _(max) nm 264.5

EXAMPLE 3

25-hydroxy vitamin D₃ -3β-0-[3-(BSA-amino) propionate] antigen and25-hydroxy vitamin D₃ antiserum:

(1) Preparation of 25-hydroxy vitamin D₃ -3β-0-[3-(BSA-amino)propionate] antigen.

25-Hydroxy vitamin D₃ -3β-0-[3-aminopropionate (18.1 mg, 38.43×10⁻ 3 mM)obtained in Example 2 was dissolved in THF (1 ml), and was added to BSA(M.W. 65,000, 50 mg, 1/50×38.43×10⁻³ mM) dissolved in tris buffer (0.1M,pH 8.6) at 0° C. with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (9.6 mg, 1.3×38.43×10⁻³ mM). Chloroform (10 ml) was addedfour times to the reaction mixture and unreacted 25-hydroxy vitamin D₃-3β-0-(3-aminopropionate ester) was removed. The aqueous layer wasfreeze dried to obtain the lyophilized product (45 mg) which was acomplex of 22 molecules of 25-hydroxy vitamin D₃-3β-0-(3-aminopropionate ester) bound through an amino group atposition-3 thereof to one molecule of BSA.

(2) Preparation of 25-hydroxy vitamin D₃ antiserum:

The lyophilized 25-hydroxy vitamin D₃ -3β-0-[3-(BSA -amino) propionate](antigen) obtained in (1) above was dissolved in tris buffer (0.1M, pH8.6). An equal amount of C.F.a. was added thereto and the material wasmixed to emulsify it so as to prepare antigen 1 82 g-200 μg/ml. Theemulsion was inoculated subcutaneously ten times each two weeks, 50γ-500γ/head, into rabbits. During this immunization, the titer of bloodsamples collected every ten days was measured and at maximum antibodytiter the whole blood was collected. The blood samples were allowed tostand at room temperature for 60 mins. to coagulate and were centrifugedat 3,000 r.p.m. for 10 mins. to obtain an antiserum containinganti-25-hydroxy vitamin D₃ antibodies, which was fractionated withammonium sulfate to collect an IgG fraction.

(3) Preparation of anti-25-hydroxy vitamin D₃ monoclonal antibodies:

Lyophilized 25-hydroxy vitamin D₃ -3β-0-[3-(BSA -amino) propionate](antigen) obtained in (1) above dissolved in phosphate buffer, pH 7.2,(50 γ) together with C.F.A. was inoculated subcutaneously into Balb/cmice, female, 4 weeks age. After 1 week, 50 γ thereof was subcutaneouslyinoculated and after 2 weeks 50 γ of antigen was intraperitoneallyinoculated. Three days after the final inoculation, their spleens werefinely cut and crushed on a mesh to prepare a spleen cell suspension.Cell fusion was performed using mouse myeloma cells P3-X-3-Ag8U1 by aconventional method. 30% PEG (M.W. 1,000) aqueous solution was incubatedat 37° C. The spleen cells hereinabove and myeloma cells (total 4×10⁷cells, 5:1) were suspended in RPMI medium (5 ml) and both cells weregently mixed, centrifuged at 1,000 r.p.m. for 10 mins., then thesupernatant was vacuum filtered. The test tube was gently shaken to mixthe cell pellets and PEG solution (1.0 ml) was slowly added and themixture was gently stirred. The mixture was incubated at 37° C. withgentle shaking, then the fusion reaction was stopped by adding slowly aconventional medium (10 ml) and again the cells were suspended. Thesuspension was centrifuged at 1,000 r.p.m. for 5 mins. The precipitatedcell pellet was dispersed by gently shaking and slowly suspended in HATmedium (5 ml) and was transferred into HAT medium (1 ml) in a vessel.The cells were observed microscopically. The cell suspension waspipetted (each 200 μl) into wells in a 96-well plate and grown in a CO₂-incubator. The fused cells were selected in HAT medium for cloning bythe limiting dilution technique. The obtained clone suspension was grownin the peritonea of pristan-treated Balb/c mice. The IgG fraction wascollected by a conventional method from ascites and serum and purifiedby affinity chromatography using protein A-bound Sepharose CL-4B. Thethus-obtained lgG subclass was IgG 1 which was used as monoclonalantibodies.

EXAMPLE 4

Preparation of radioisotope monoiodine [¹²⁵ I]-labeled 25-hydroxyvitamin D₃ derivative; 25-hydroxy vitamin D₃-3β-0-{3-[N-3-(4-hydroxy-3-iodo [¹²⁵ I] phenyl) propionyl] aminopropionate}:

¹²⁵ I] Bolton-Hunter reagent [NEN, NEX-120-10, 2200 Ci/mM, Total 0.33mCi/100 μl benzene solution] (100 pM/66.6 μl/220 μCi) and DMAP (1 nM/1μl THF) were added to a THF solution (130 μl) of 25-hydroxy vitamin D₃-3β-0-(3-aminopropionate) (100 nM/127.8 μl THF) obtained in Example 2,and the mixture was reacted at room temperature for 24 hours under anargon atmosphere in the dark. The reaction mixture was purified by HPLC(Zorbax-SIL, 4.6 mm×25 cm column 20% isopropanol-n-hexane, flow 1ml/min.) The compound hereinabove has Rt=8.50-9.50 min. and recovery20.0%. (Iodine [¹²⁵ I] Bolton-Hunter reagent: Rt=14.0-16.0 min.)Radioisotope monoiodine-labeled 25-hydroxy vitamin D₃ was identifiedwith synthesized non-radioisotope monoiodine-labeled 25-hydroxy vitaminD₃ by HPLC.

EXAMPLE 5

Preparation of radioisotope diiodine [¹²⁵ I]-labeled 25-hydroxy vitaminD₃ derivative; 25-hydroxy vitamin D₃ -3β-0-{3-[N-3,5-diiodo-4-hydroxy[¹²⁵ I] phenyl)propionyl] amino propionate}:

Iodine [¹²⁵ I] Bolton-Hunter reagent [NEN, NEX-120H -10, 4400 Ci/mM,Total 0.33 mCi/100 μl benzene solution] (25 pM/33.3 μl/110 μCi) and DMAP(1 nM/1μl THF) were added to THF solution (120 μl) of 25-hydroxy vitaminD₃ -3β-0-(3-aminopropionate) (25 nM/6.952 μl THF) obtained in Example 2,and the mixture was reacted at room temperature for 20 hours under anargon atmosphere in the dark. The reaction mixture was purified by HPLC(Zorbax-SIL, 4.6 mm×25 cm column, 20% isopropanol-n-hexane, flow 1ml/min.) The compound hereinabove has Rt=8.90-10.10 min. and recovery9.9%. (Iodine [¹²⁵ I] Bolton-Hunter reagent: Rt=14.0-16.0 min.)Radioisotope diiodine-labeled 25-hydroxy vitamin D₃ was identified withsynthesized non-radioisotope diiodine labeled 25-hydroxy vitamin D₃ byHPLC.

EXAMPLE 6

Evaluation of radioisotope iodine [¹²⁵ I]-labeled 25-hydroxy vitamin D₃derivative:

(1) Preparation of standard curve using radio immunoassay of 25-hydroxyvitamin D_(3:)

A standard ethanol solution of 25-hydroxy vitamin D₃ (256 pg/20 μl) wasdiluted stepwise by two-fold dilution to prepare 128 pg/20 μl, 64 pg/μl,32 pg/20 μl, 16pg/20 μl, 8pg/20 μl, 4 pg/20 μl and 2 pg/20 μl samples.

Samples (20 μl) of each concentration hereinabove were pipetted intothree tubes. Ethanol solutions, each 20 μl, of radioisotope iodine [¹²⁵I]-labeled 25-hydroxy vitamin D₃ derivative which contains as anantioxidant tocopherol was pipetted into each tube (20 μl, ca. 13,000cpm).

Anti-25-hydroxy vitamin D₃ rabbit serum diluted with tris buffer(approx. 15,000 dilution) pH 8.6, each 1 ml, was added to each tube.Each mixture in the tubes was stirred in a BORTEX mixer and allowed tostand at 5° C. overnight (20-24 hours). Glycine buffer (pH 8.6)suspending a mixture of dextran (T70) and active charcoal (1:10), each300 μl) was added into the reaction tubes, and each was stirred by aBORTEX mixer and incubated at 5° C. for 1 hour. B-F separation wasperformed by centrifugation at 3,000 r.p.m. for 10 mins., and eachsupernatant solution (1 ml) was measured by an Autowell γ-counter for Imin. The ratio of binding is calculated by the following equation:##EQU1## wherein (B) is the count number in each tube, (BL) is the countnumber measured by using 25-hydroxy vitamin D₃ (1,000 pg/20 μl) in placeof isotope ¹²⁵ I-labeled 25-hydroxy vitamin D₃ derivative, and (Bo) isthe count number measured by using 25-hydroxy vitamin D₃ at zeroconcentration. FIG. 1 shows the standard curve of [¹²⁵ I]-labeled25-hydroxy vitamin D₃ derivative.

(2) Comparison between tritium [³ H]-labeled and ¹²⁵ I]-labeled25-hydroxy vitamin D₃ derivative:

A standard curve was prepared by using tritium [3H]-labeled 25-hydroxyvitamin D₃ derivative (26, 27-methy-³ H) as in the case of the [¹²⁵I]-labeled derivative in (1) hereinabove.

The results are shown in FIG. 2. In FIG. 2, -- -- --: [¹²⁵ I]-labeledand -- -- --: [³ H]-labeled compounds were used. Equal or highersensitivity was found when using [¹²⁵ I] labeling as compared with [³ H]labeling, especially at low concentrations (10 pg/Tube), and highersensitivity was observed.

(3) Comparison between monoiodine [¹²⁵ I] labeling and diiodine [¹²⁵ I]labeling:

Monoiodine [¹²⁵ I] labeling and diiodine [¹²⁵ I] labeling were comparedaccording to the same method as hereinabove. The results are shown inFIG. 3. In FIG. 3, -- -- -- indicates monoiodine [¹²⁵ I] labeling, and-- -- -- indicates diiodine [¹²⁵ I] labeling.

A radioisotope iodine-labeled 25-hydroxy vitamin D₃ derivative isillustrated in formula [V] ##STR5## Assays using monoiodine [¹²⁵I]-labeled compound and diiodine [¹²⁵ I]-labeled compound show almostthe same sensitivity, but monoiodine [¹²⁵ I] labeling is preferred.

(4) Comparison with methylene chain length in R_(1:)

In formula [IV], assays using compounds having methylene R₁ [--CH₂)n--]wherein n=1, n=2 and n=3, are compared with each other.

The results are shown in FIG. 4, in which -- -- -- indicates n=1, -- ---- indicates n=2 and -- -- -- indicates n=3. It will be seen that n=2and n=3 show good results as compared with n=1. Particularly preferredis n=2.

EXAMPLE 7

Evaluation of radioisotope iodine-labeled 25-hydroxy vitamin D₃ by theCPBA method using DBP in place of anti-25-hydroxy vitamin D₃ antibodies:

(1) Preparation of DBP:

Fetal calf serum (FCS) (3.3 ml) diluted with phosphate buffer (pH 7.4,0.01M) was charged on a column of Blue-Sepharose (tradename PharmaciaCorp. φ2.5×7.5 cm) and a de-albumination treatment through a gel-bedvolume of 40 ml was performed.

Fractions, each 5 ml, Nos. 7-10, with absorption at 280 nm, werecollected (total 20 ml). Alternatively, FCS can be replaced by the serumof homeothermal animals.

(2) The standard curve of radioisotope iodine [¹²⁵ I]-labeled 25-hydroxyvitamin D₃ by the CPBA method using DBP, instead of antibodies, isplotted as follows:

A standard ethanol solution of 25-hydroxy vitamin D₃ (1,024 pg/20 μl)was diluted stepwise by two-fold dilutions to prepare 32 pg/20 μl, 16pg/20 μl, 8 pg/20 μl, 4 pg/20 μl, 2 pg/20 μl and 1 pg/20 μl.

Samples (20 μl) of each concentration hereinabove were pipetted into twotubes. Aliquots of an ethanol solution, each 20 μl, of radioisotopeiodine [¹²⁵ I]-labeled 25-hydroxy vitamin D₃ derivative, containing asan antioxidant tocopherol, were pipetted into each tube (20 μl, ca.30,000 cpm). DBP diluted with tris buffer, pH 8.6 (approx. 500-1,000dilution of DBP fraction in (1) hereinabove) each 500 μl, was stirredand allowed to stand at 4° C. for 16 hours.

Glycine buffer, pH 8.6, suspending a mixture of dextran (T70) and activecharcoal (1:10) (each 30 μl), was added into the reaction tubes, stirredand incubated at 4° C. for 30 mins. B-F separation was made bycentrifugation at 3,000 r.p.m. for 10 mins. at 4° C., and eachsupernatant solution 500 μl was measured by an Autowell γ-counter for 1min. The ratio of binding is calculated by the following equation:##EQU2## wherein (B) is the count number in each tube, (NSB)₋₋ is thecount number measured by using 25-hydroxy vitamin D₃ (1,024 pg/20 μl)instead of ¹² 5I-labeled 25-hydroxy vitamin D₃ derivative and (Bo) isthe count number measured by using 25-hydroxy vitamin D₃ at zeroconcentration. FIG. 5 shows the standard curve of [¹²⁵ I]-labeled25-hydroxy vitamin D₃ derivative.

(3) Stability of radioisotope iodine [¹²⁵ I]-labeled 25-hydroxy vitaminD₃ derivative:

A 50% H20-ethanol solution of [¹²⁵ I] 25-hydroxy vitamin D₃ derivativeobtained in Example 4 or Example 5 was stored at -20° C., in an argonatmosphere in a vial. A standard curve using the above sample wasprepared according to method (2) hereinabove. In FIG. 6, --◯--◯--indicates day 0, --Δ--Δ-- indicating day 21 and --□--□-- indicating day49 of the standard curve are shown. As is shown in FIG. 6, when thematerial was stored at -20° C., no changes of the standard curve withina range of 2-32 pg/Test were observed. The results show that the [¹²⁵ I]25-hydroxy vitamin D₃ derivative can be used in radio immunoassay withgood stability.

It will thus be seen that the present invention provides radioisotopeiodine-labeled 25-hydroxy vitamin D₃ derivatives with high radiationenergy and wide usability, compared to low radiation energy, β-rayemitting tritium [³ H]-labeled vitamin D₃. It might be though that highradiation energy radioisotope iodine [¹²⁵ I] vitamin D₃ would degradeautomatically due to reaction on the conjugated double bond in thevitamin D structure. But [¹²⁵ I]-labeled 25-hydroxy vitamin D₃derivative of the present invention in fact does not autodegrade byγ-rays and is quite stable for use in radio immunoassay.

In general, vitamin D₃ is metabolized in vivo in the liver or kidney andis converted to activated vitamin D₃. The activated vitamin D₃, such as25-hydroxy vitamin D₃, 1α, 25-dihydroxy vitamin D₃, 1α-hydroxy vitaminD₃ and 1α,24-dihydroxy vitamin D₃, is used clinically for the therapy ofosteoporosis and osteomalacia. The clinical dose thereof is quite lowdue to its strong physicological action. The pharmacological activity iscorrelated to the blood level and tissue level of the drug and hencemeasurement of the blood level of the drug administered to humans isimportant from a clinical point of view. Considering the above, thepresent invention is valuable for the assay of activated vitamin D₃ inclinical tests.

What is claimed is:
 1. A 25-hydroxy vitamin D₃ amino acid of the formula##STR6## wherein R₁ is --(CH₂)_(n--), wherein n is an integer from 1 to10.
 2. A derivative according to claim 1, wherein n is an integer from 2to
 6. 3. A process for the production of a 25-hydroxy vitamin D₃ aminoacid derivative of the formula ##STR7## wherein R₁ is --(CH₂)_(n--),wherein n is an integer from 1 to 10, comprising reacting 25-hydroxyvitamin D₃ with an amino acid having a protected amino group to form a25-hydroxy vitamin D₃ derivative of the formula ##STR8## wherein R₁ is--(CH₂)_(n--), wherein n is an integer from 1 to 10, and R₂ is aprotective group for the amino group; and removing said protective groupin the presence of a base in an inert solvent.
 4. A process according toclaim 3 wherein the protective group for the amino group is9-fluorenylmethyloxycarbonyl.
 5. The process according to claim 3,wherein n is an integer from 2 to 6.